Accomplishments for FY2009: 1) We have shown for the first time that mammalian prions that cause a transmissible spongiform encephalopathy (TSE or prion disease) can be generated solely from bacterially expressed recombinant prion protein. This discovery provides the most compelling evidence so far for the potential protein-only prion hypothesis for the nature of the TSE infectious agent. However, the data leave open the likelihood that other molecules strongly enhance the infectious titers of pathological forms of prion protein. 2) We have extended our characterization of the structure of the flexible amino-terminal domain of PrP when bound to pentosan polysulfate, one of the most potent known anti-TSE therapeutic compounds. Nuclear magnetic resonance, fluorescence and circular dichroism experiments have refined our new three dimensional structure of the PrP:pentosan polysulfate complex. 3)We have refined our understanding of the smallest and most infectious prion particles using improved field-flow fractionation and prion detection methodologies. 4) We have pursued new methods for characterizing and visualizing the pathogenic form of prion protein when bound to membranes. 5) We have detected TSE strain-dependent differences in the interactions between prion protein and C1q, a complement factor. 6) We have use hydrogen-deuterium exchange and mass spectrometry to determined that infectious prion-seeded amyloid fibrils of bacterially expressed recombinant prion protein have a different structure than spontaneously formed (unseeded) fibrils. This work provided strong biochemical evidence of the distinct seeding capacity of infectious prions.